Enzymes_Industrial Microbiology_Postgraduate.pptx

Enzymes_Industrial Microbiology_Postgraduate

1. Enzymes Generation of industrial microbial products

2. Introduction Enzymes are organic compounds mainly proteins, which catalyze all the chemical reactions of living things – plants, animals, and microorganisms. Enzymes can act on their own after extraction from sources. Most industrial enzymes are obtained from microorganisms because- Microorganisms grow faster . Cost-effective as small land and can be produced in any desired amount. Diversity of microbes helps to produce enzymes that can operate under extreme environmental conditions . For example, amylases can function at 110° C, and proteases can operate at pH values as high as 11 or as low as 3. Microorganisms can be manipulated to produce desired enzymes.

3. Mechanism of Enzyme

4. Classification of Enzymes

5. The techniques used for microbial production of enzymes are described as; Isolation and selection of microbial strains. Formulation of medium Fermentation Extraction and purification of enzymes.

6. A. Isolation and selection of microbial strains Important criteria for selecting microbes are: To produce high amount of enzymes Less fermentation time Simple isolation and separation Non-pathogenic cell and Utilization of low cost culture medium Strain improvement techniques are used production by the mutagens or UV rays method .

7. B. Formulation of medium T he cu l t ure m ed i um s h o uld con t ain all th e n u t rie n t s t o s upp o rt a d eq u ate gr o w th of microorganism that will result in maximum enzyme production . All ingredients should be easily available at low cost and with high nutritional value. The producti o n m edia m ust co n t a i n so u rces o f car b on, n i trog e n, e ne r g y , m i n era l s, macronutrients, micronutrients, growth factors, etc . Commonly used substrate for the media are soyabean meal, casein, yeast extract etc. The pH of medium should be adjusted to show optimal microbial growth and enzymes production.

8. C. Fermentation In Biotechnology, Fermentation means any process by which microorganisms are grown in large quantities to produce any type of useful materials . Submer ged cul t u r e tec h nique is c om m on ly us e d for in d u s tri a l pro d u c t io n of enzymes . Submerged culture technique is most widely used because of less chances of infection and it give more yield. Surface culture technique is traditional method but still in use for production of fungal enzyme such as amylase, proteases, cellulose and pectinases .

9. Mechanically stirred capacities 20,000 to 1,00,000 liters are used for production of enzymes by submerged techniques . Duration is 2 to 7 days . The growth condition such as pH, temp and oxygen are maintained in optimum level . A small amount of oil is added to control the foam in fermentation . Most of enzymes are extracellular and produced at the end of exponential phase . Solid substrate culture method is mainly used for isolation of enzymes from fungi.

10. D. Extraction and purification of enzymes The desired enzyme produced may be extracted into the culture medium which are based on intracellular or extracellular nature of isolate. The physical, chemical and enzymatic methods are used to break the cells an d release the intracellular enzyme. Recovery of extracellular enzyme is more simple than intracellular enzyme . Fungal broth is directly filtered or centrifuged after pH adjustment . Bacterial broth is first treat with calcium salts to precipitate calcium phosphate which help in separation of bacterial cells and colloids.

11. Isolated enzymes can be precipitated by using salts and organic solvents. Chromatographic techniques such as ion-exchange, size exclusion, affinity, hydrophobic interaction and dye ligand are commonly used for separation and purification of enzymes.

12. Submerged fermentations, predominantly produce microbial enzymes, Solid-substrate fermentations are used, particularly for producing extracellular fungal enzymes. Most industrial enzymes are products of batch processes and few are currently produced via continuous fermentation . Enzyme Production Process

13. Submerged fermentation is a method of manufacturing biomolecules in which the growth of microorganisms depends on nutrients provided by liquid substrates (molasses, broths). Inoculum (bacteria or fungi) is added in a small closed flask containing the rich nutrient broth. A high volume of oxygen is also required for the process. The production of enzymes occurs when the microorganisms interact with the nutrients of broth. The bioactive compounds are secreted into the fermentation broth when they are extracellular. 1. Submerged Fermentation

14. Submerged Fermentation

15. Mostly used in pharmaceuticals, food, textiles etc. SSF is a method to produce metabolites of microorganisms using solid support in place of microbes without free-flowing aqueous phase. The support used is especially grain brans (rice, wheat), de-oiled oil seed cakes, and other similar substances. Substances are acidified with HCl, mineral salts . Inducers such as 10% starch for amylase , and gelatin and pectin for protein and pectinase are used. Mostly fungi are used as inoculum because of their capability to produce high levels of enzymes in low moisture conditions and higher degree of aeration. 2. Solid State Fermentation (SSF)

16. Fungi are inoculated in moist bran and placed in flat trays or a revolving drum. Moisture (about 8%) is maintained by spraying water on the trays and by circulating moist air over the preparation The temperature of the bran is kept at about 30° C by the circulating cool air The production period is usually 30-40 hours but could be as long as seven days. Enzymes are preserved by drying at about 37° C – 40° C and ground SSF Process

17. SSF Fermenter

18. Bulk Industrial Enzyme Production Overview

19. PROPERTIES BACTERIAL ENZYMES

20. Industrial Enzymes 1. TEXTILE INDUSTRY-

21. 2. DETERGENT INDUSTRY-

22. 3. PULP AND PAPER INDUSTRY-

23. 4. LEATHER INDUSTRY-

24. AMYLASE Hydrolyses alpha-1,4-glucosidic bonds to reduce viscosity of gelatinized starch, producing soluble dextrin. GLUCOAMYLASE To saccharify liquified starch from various sources such as corn, wheat . Resultant are glucose rich syrups. DEXTRANASE FOR SUGAR INDUSTRY Dextran are undesirable compounds in sugar production which reduces viscosity and reduces industrial recovery. GLUCOSE ISOMERASE Catalyses isomerization of glucose to fructose. 5. STARCH AND SUGAR INDUSTRY -

25. 6. DAIRY INDUSTRY- AMYLASE For starch modification XYLANASE To break down Xylan. 7. BAKING INDUSTRY - 8. ANIMAL FEED INDUSTRY -

26. Amylase

27. Amylase catalyzes the hydrolysis of starch into sugars, glycogen , and related polysaccharides. Starch is a polysaccharide composed of two types of polymers; amylose (linear α- 1,4-linked glucose units constituting 20-30%) and amylopectin (branched polymer with both α -1,4 and α- 1,6 linkages constituting 75-80%). It can be found in animals, plants, and microbes. Introduction

28. Different amylases attack starch differently. Amylases are classified into three major categories. α- amylase An endo- 1,4-α- D-glucan glucanohydrolase enzyme. Catalyzes the hydrolysis of internal α- -1,4 glycosidic linkages randomly to glucose, maltose and maltotriose units. Activity depends on the presence of metal co-factor as it is a calcium metalloenzyme. Optimum pH for activity is 7. β - amylase An exo - 1,4- α -D-glucan maltohydrolase enzyme. Acts on the non-reducing end of polysaccharide and hydrolyze α--1,4 glycosidic linkages sequentially to maltose or β -dextrin Can’t cleave branched linkages in branched polysaccharides; glycogen or amylopectin. Optimum pH- 4-5.5 Involved in germination and food ripening. γ – amylase A glucosidase; cleaves both α -1,6 glycosidic linkages and the last α--1,4 glycosidic linkages at the non-reducing end of amylose and amylopectin, yielding glucose. Optimum pH- 3 Types of Amylase

29. Scheme for the hydrolysis of starch by amylase

30. Sources - Maize, cassava, sorghum, potatoes, and industrial residues generated during the production of starch products. Microorganisms involved in starch fermentation process- Production of Amylase Bacteria Fungi Bacillus subtilis Aspergillus niger B. amyloiquefaciens Penicillium sp. B. licheniformis P. brunneum B. stearothermophilus P. fellutanum B. coagulans

31. 3 . Fermentation process- Solid state fermentation ( nutrient-rich waste material can be used as substrate) Submerged fermentation 4. Process parameters on the microbial source, desired end product, method of fermentation employed, etc. Media Temperature pH Duration of fermentation Carbon source- Maltose, sucrose, glucose, wheat bran, gram husk, rice bran and mustard oilseed cake Aspergillus – 30-90 C Aspergillus – 5.0-6.0 Aspergillus – 96 hour Nitrogen source- Inorganic nitrogen sources ammonium sulfate, NaCl, ( NH 4 ) 2 HPO . Organic source- Peptone, yeast extract, and soybean meat. Penicillium- 30- 55 C Penicillium- 6.0-7.0 Penicillium- 72 hour Bacterial amylase- 55 C Bacteria- Near 7.0 Bacteria- Approximately 6 days

32. 5. Recovery and Purification of Enzymes: Enzymes can be either extracellular or intracellular, but most of the industrial enzymes are extracellular that means they remain in the medium after removing biomass. If the enzymes are intracellular, special techniques are needed for cell disruption. Sonication , high-pressure, glass beads can be used for disruption of cells. Removal of cell debris: Filtration or centrifugation Removal of nucleic acids: Precipitation Enzyme precipitation: Salts (ammonium sulfate), organic solvents (isopropanol, ethanol, and acetone) Liquid-liquid partition: Concentrated enzymes are achieved. Separation by Chromatography: Ion exchange, size exclusion, affinity, hydrophobic interaction, etc. Drying and packaging.

33. Application of amylase α amylase- Starch processing — Liquefaction of starch in the production of sugar syrups. Baking — Partial starch degradation in flour modification and the generation of fermentable sugars, and for improved crust color. Brewing — Starch hydrolysis during wort preparation. Biological detergents — Starch removal from food stains. Textiles manufacture — Desizing β-Amylase- Maltose syrup production. Brewing— Increasing wort fermentability.

34. Protease

35. Proteases are the first enzymes to be commercialized and are responsible for the breakthrough in industrial enzyme development. A protease (also called peptidase, proteinase, or peptide hydrolase ) is an enzyme that catalyzes proteolysis , the breakdown of proteins through the hydrolysis of peptide bonds into smaller polypeptides, oligopeptides, or single amino acids. Introduction Protease

36. Types Based on s ite of cleavage Exopeptidases - act on the terminus of polypeptide chain Endopeptidases - act internally in polypeptide chains Based on o ptimal pH Acid protease Basic protease Neutral protease Based on m echanism of action Serine protease Threonine protease Metalloprotease Glutamic acid protease

37. Media formulation: The induction of protease requires a substrate like peptone, casein, and other proteins. Agricultural wastes like rice bran, paddy straw, and pigeon pea waste are also used for protease production. Nitrogen sources- soybean, milk, casein, gelatin etc. Carbohydrate source- starch, molasses, barley, corn, wheat etc. Production Process of Protease

38. 2. Microorganisms: Bacteria Fungi Bacillus subtilis Aspergillus flavus Bacillus pumilus Aspergillus fumigatus Bacillus licheniformis Aspergillus melleus Bacillus altitudinis Aspergillus niger Bacillus firmus Fusarium graminearum Bacillus amyloliquefaciens Penicillium griseofulvim Bacillus proteolyticus Penicillium lilacinus Thermomonospora fusca

39. 3. Fermentation: For bacteria cultivation, submerged fermentation is preferable. For fungi , solid-state fermentation is more suitable. 4. Protease Recovery: Filtration and centrifugation for insoluble product removal. Biomass is treated with to destabilize microorganisms Evaporation, membrane filtration, or crystallization for concentrating protease enzymes in broth Drying and packaging

40. Dairy industry: For cheese manufacturing, protease hydrolyzes specific peptide bond ( Phe105 - Met106 bond) to generate para casein and macro peptides. Baking industry: Generate better texture and crust color for bread. Protease helps in bakery industry by degrading and decreasing gluten content. Brewing industry: B. subtilis protease solubilizes proteins from barely for wort production. Microbial proteases hydrolyze proteinaceous substances. Meat tenderization: Bromelain, papain, ficin, and collagenase are widely used to improve meat tenderness. Industrial production of Aspartame: It is a synthetic non-carbohydrate, zero-calorie sweetener that is synthesized by proteases. Detergent: Proteases hydrolyze the peptide bonds of proteins staining the fabric. Fabrics industry : Protein fabrics such as wool and silk are treated by a protease to break down the fibrils on the surface, so the look and feel of the fabric softens. Applications of Protease

41. Lipase

42. Lipases are serine hydrolases that catalyze the hydrolysis of ester bonds in long-chain triacylglycerols with a concomitant release of fatty acids and glycerol. Lipases are highly specific as chemo-, regio-, and enantioselective biocatalysts. Under micro-aqueous conditions lipase shows reverse reactions, resulting in esterification, alcoholysis, and acidolysis. They are extracellular enzymes. Introduction

43. Sources: Natural oils, mustard oil cake, neem oil cake, groundnut oil cake, olive oil, synthetic triglycerides, and esters of fatty acids. Rice bran, wheat bran, almond meal. 2. Microorganisms: Isolation, screening and culturing of lipase-producing microbes. Production Process Bacteria Fungi Pseudomonas mendocina Candida lipolytica Pseudomonas aeruginosa Candida antarctica Fervidobacterium changbaicum Aspergillus niger Fervidobacterium nodosum Aspergillus oryzae Bacillus subtilis - Staphylococcus -

44. 3. Fermentation: Submerged fermentation (mostly used) Solid-state fermentation 4. Optimum condition for Fermentation (for bacteria): pH – 7-10 Temperature – 30-60 C Incubation period – 1-5 days 5. Purification / Harvesting: CaCl 2 precipitation: This is done for fatty acid precipitation followed by centrifugation. Then, the supernatant is treated with chilled acetone for protein precipitation followed by centrifugation and ultrafiltration. ( NH 4 ) 2 SO 4 precipitation Coloumn chromatography

45. Industrial application of lipase: Applications of Lipase

46. 2. Lipase in detergents: Due to the capability of hydrolysis of fat and lipids, lipase is used in laundry industries and household detergent. Laundry detergent instructs flexibility to fabrics, softness, and dissolved in water. 3. Pulp and paper industry: Pitch is the insoluble component of wood (mostly waxes, and triglycerides), which causes drastic troubles in pulp and paper manufacturing but can be removed using lipase. 4. Lipase in biofuels production: Biodiesel (fatty acid methyl ester) derived from the transesterification of triglycerides is emerging as a viable alternative to replace petroleum-based diesel. Transesterification is the displacement of alcohol from an ester by another alcohol, which has been used as an alternative process to improve the physical properties of biodiesel. Instead of using refined oil transesterification from sunflower frying oil is an economical alternative because of its low cost. Lipase is used as a catalyst for transesterification of waste sunflower frying oil.

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